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The impact of DNA demethylation on the upregulation of the NRN1 and TNFAIP3 genes associated with advanced gastric cancer

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Abstract

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide. Very few therapeutic options are currently available in this neoplasia. The use of 5-Aza-2′-deoxycytidine (5-AZAdC) was approved for the treatment of myelodysplastic syndromes, and this drug can treat solid tumours at low doses. Epigenetic manipulation of GC cell lines is a useful tool to better understand gene expression regulatory mechanisms for clinical applications. Therefore, we compared the gene expression profile of 5-AZAdC-treated and untreated GC cell lines by a microarray assay. Among the genes identified in this analysis, we selected NRN1 and TNFAIP3 to be evaluated for gene expression by RT-qPCR and DNA methylation by bisulfite DNA next-generation sequencing in 43 and 52 pairs of GC and adjacent non-neoplastic tissue samples, respectively. We identified 83 candidate genes modulated by DNA methylation in GC cell lines. Increased expression of NRN1 and TNFAIP3 was associated with advanced tumours (P < 0.05). We showed that increased NRN1 and TNFAIP3 expression seems to be regulated by DNA demethylation in GC samples: inverse correlations between the mRNA and DNA methylation levels in the promoter of NRN1 (P < 0.05) and the intron of TNFAIP3 (P < 0.05) were detected. Reduced NRN1 promoter methylation was associated with III/IV TNM stage tumours (P = 0.03) and the presence of Helicobacter pylori infection (P = 0.02). The identification of demethylated activated genes in GC may be useful in clinical practice, stratifying patients who are less likely to benefit from 5-AZAdC-based therapies.

Key messages

  • Higher expression of NRN1 and TNFAIP3 is associated with advanced gastric cancer (GC).

  • NRN1 promoter hypomethylation contributes to gene upregulation in advanced GC.

  • TNFAIP3 intronic-specific CpG site demethylation contributes to gene upregulation in GC.

  • These findings may be useful to stratify GC patients who are less likely to benefit from DNA demethylating-based therapies.

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Acknowledgments

We are grateful to Brunno dos Santos Pereira, Renata Sanches de Almeida, and Camila Albuquerque Pinto for the help in sample collection and to Brunno dos Santos Pereira for the help in generating the final figures.

Funding

This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; to FW, DQC, JCG, MFL, and MACS), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; to FW, DQC, JCG, RRB, and MACS), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; to COG and ACA).

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Author notes

  1. Fernanda Wisnieski, Leonardo Caires Santos and Danielle Queiroz Calcagno contributed equally to this work.

Authors and Affiliations

  1. Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, Rua Botucatu, 740, São Paulo, São Paulo, 04023900, Brazil

    Fernanda Wisnieski, Leonardo Caires Santos, Jaqueline Cruz Geraldis, Ana Carolina Anauate, Elizabeth Suchi Chen, Mariana Ferreira Leal & Marilia Arruda Cardoso Smith

  2. Disciplina de Gastroenterologia, Departamento de Medicina, Universidade Federal de São Paulo, Rua Loefgreen, 1726, São Paulo, São Paulo, 04040002, Brazil

    Fernanda Wisnieski

  3. Programa de Pós-graduação em Oncologia e Ciências Médicas, Universidade Federal do Pará, Rua dos Mundurucus, 4487, Belém, Pará, 66073-000, Brazil

    Danielle Queiroz Calcagno, Samia Demachki, Paulo Pimentel Assumpção, Mariana Ferreira Leal & Rommel Rodriguez Burbano

  4. Departamento de Patologia, Universidade Federal de São Paulo, Rua Botucatu, 740, São Paulo, São Paulo, 04023900, Brazil

    Carolina Oliveira Gigek & Ricardo Artigiani

  5. Disciplina de Genética, Hemocentro da Faculdade de Medicina de Marília, Rua Lourival Freire, 240, Marília, São Paulo, 17519-050, Brazil

    Lucas Trevizani Rasmussen & Spencer Luiz Marques Payão

  6. Disciplina de Gastroenterologia Cirúrgica, Departamento de Cirurgia, Universidade Federal de São Paulo, R. Napoleão de Barros, 715, São Paulo, 04024002, Brazil

    Laercio Gomes Lourenço & Carlos Haruo Arasaki

  7. Austrian Institute of Technology, Center for Health & Bioresources, Molecular Diagnostics, Giefinggasse 4, 1210, Vienna, Austria

    Stephan Pabinger & Julie Krainer

  8. Laboratório de Biologia Molecular, Hospital Ophir Loyola, Avenida Governador Magalhães, 992, Belém, 66063-240, Brazil

    Rommel Rodriguez Burbano

Authors
  1. Fernanda Wisnieski
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  2. Leonardo Caires Santos
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  3. Danielle Queiroz Calcagno
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  4. Jaqueline Cruz Geraldis
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  5. Carolina Oliveira Gigek
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  19. Marilia Arruda Cardoso Smith
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Contributions

Conception and design: FW, MFL, RRB, and MACS. In vitro experiments: FW and DQ. Sample collection: FW, LCS, DQC, JCG, COG, ACA, ESC, PPA, LGL, and CHA. Pathological analysis: RA and SD. H. pylori diagnosis: LTR and SLMP. Molecular experiments: FW, LCS, DQC, JCG, COG, ACA, ESC, and MFL. Data analysis: FW, LCS, JCG, SP, and JK. Writing, review, and/or revision of the manuscript: all authors. Administrative, technical, or material support: MACS and RRB.

Corresponding authors

Correspondence to Fernanda Wisnieski or Marilia Arruda Cardoso Smith.

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The study was approved by medical ethics committee of each study center. Written informed consent was obtained from each patient.

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Supplementary Fig. 1

GC cell line viability after 5-AZAdC treatment, Each point represents the mean ± standard error of three independent experiments. (PNG 1376 kb)

High resolution image (TIF 353 kb)

Supplementary Table 1

Primer sequences for DNA methylation analysis, *For target amplification, 25 ng of bisulfite-treated DNA was used in PCR reactions with Platinum™ Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) (XLSX 10 kb)

Supplementary Table 2

List of 83 DEGs by comparing 5-AZAdC-treated and non-treated GC cell lines using de RP analysis. FC: fold change, FDR: false discovery rate (XLSX 12 kb)

Supplementary Table 3

Clinicopathological associations of NRN1 mRNA and DNA methylation levels. aAccording to the Lauren classification [9], bAccording to AJCC [10], c, dDetected in tumour samples as described previously [8], *Significant difference between groups by the Mann-Whitney test, n: number of samples. IQR: interquartile range (XLSX 13 kb)

Supplementary Table 4

Clinicopathological associations of TNFAIP3 mRNA and DNA methylation levels, aAccording to the Lauren classification [9], bAccording to AJCC [10], c, dDetected in tumour samples as described previously [8], *Significant difference between groups by Mann-Whitney test, n: number of samples. IQR: interquartile range (XLSX 15 kb)

Supplementary Table 5

Correlation between NRN1 mRNA and methylation levels in gastric tissue samples. (XLSX 13 kb)

Supplementary Table 6

Correlation between TNFAIP3 mRNA and methylation levels in GC tissue samples. (XLSX 15 kb)

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Wisnieski, F., Santos, L.C., Calcagno, D.Q. et al. The impact of DNA demethylation on the upregulation of the NRN1 and TNFAIP3 genes associated with advanced gastric cancer. J Mol Med 98, 707–717 (2020). https://doi.org/10.1007/s00109-020-01902-1

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